Project Summary/Abstract Recent clinical trials and a host of nonhuman primate studies have pushed the HIV vaccine field further toward the development of concepts based on humoral anti-HIV envelope immunity. However, such efforts face a major hurdle in that the routinely short-lived humoral responses to HIV envelope antigens will severely compromise HIV vaccine efficacy. An additional caveat is that any solution to the humoral persistence problem must include balanced immune responses that do not engender forms of CD4+ T cell activation that might increase HIV infection risk. The recent RV144 clinical trial of a poxvirus/ envelope protein regimen underscores the issue in humans. In this trial, anti-gp120 antibody responses, including those linked with partial efficacy, decayed to undetectable levels within 12 months. Based on the available literature, we hypothesize that anti-gp120 titers rapidly fade in this manner because 1) HIV gp120-specific plasma cells are extensively killed during the germinal center reaction or 2) HIV gp120-specific plasma cells survive the germinal center reaction but cannot move into the bone marrow to become long-lived cells. Theoretically, these questions can be addressed by comparative analyses of vaccine regimens that yield persistent antibody titers versus ones that do not. This strategy has been hampered by a paucity of regimens that reliably yield persistent anti-gp120 humoral responses. Our group has tested the same gp120-based antigen in a wide variety of adjuvant formulations and vaccination regimens in macaques. Recently we determined that one of these regimens, which involves coimmunizing with envelope protein in adjuvant along with electroporation of DNA encoding IL-12 (herein termed DNA/protein coimmunization), generates significantly more persistent antibody titers compared to any other approach we tested. These findings now provide the necessary means to conduct comparative studies that elucidate the determinants for persistent anti-gp120 antibody responses. Accordingly, this Project will juxtapose the DNA/protein coimmunization regimen with a matched protein/adjuvant regimen that yields non-persistent responses. Our proposed comparative analyses will be driven by the two hypotheses listed above. In two specific aims, we will address whether persistent anti-HIV antibody responses are distinguished by 1) the preservation of short-term anti-gp120 plasmablasts without the enhancement of long-lived plasma cells in the bone marrow or 2) the establishment of long-lived anti-gp120 plasma cells in the bone marrow. We will also exploit the multifaceted nature of DNA/protein coimmunization to Identify vaccine components and associated immunological pathways that afford persistent humoral anti-gp120 responses, desirable CD4+ T cell profiles and protective efficacy against heterologous SHIV challenge. Information from this project should have significant impact, providing broadly applicable, immune-based underpinnings for the development of vaccine regimens that provide persistent anti-envelope antibody responses.